1. Field of the Invention
The present invention is directed to methods of assessment of gene expression, and more particularly to methods of assessment of gene expression that utilize multi-targeted priming for genome-wide gene expression assays.
2. Description of the Related Art
Gene expression levels have been quantified by numerous procedures, including reverse transcription (RT)-PCR (Bustin S A, J Mol Endocrinol 2000, 25(2):169-193), sequencing of expressed sequence tags (ESTs) (Adams M D, Kelley J M, Gocayne J D, Dubnick M, Polymeropoulos M H, Xiao H, Merril C R, Wu A, Olde B, Moreno R F, Science 1991, 252(5013):1651-1656), serial analysis of gene expression (SAGE) (Velculescu V E, Zhang L, Vogelstein B, Kinzler K W, Science 1995, 270(5235):4840, microarray hybridization (DeRisi J L, Iyer V R, Brown P O, Science 1997, 278(5338):680), and massively parallel signature sequencing (Nagalakshmi U, Wang Z, Waern K, Shou C, Raha D, Gerstein M, Snyder M, Science 2008, 320(5881):1344). Rapid development of platforms has improved throughput, but also generated strong demand for enhanced sensitivity and measurement accuracy. For nearly all expression assays, reverse transcription from messenger RNA (mRNA) to complementary DNA (cDNA) is a key step of the process that contributes less experimental variance than biological growth and harvest, but greater experimental variance than hybridization (Yang Y H, Speed T, Nat Rev Genet 2002, 3(8):579-588 and Wernisch L, Kendall S L, Soneji S, Wietzorrek A, Parish T, Hinds J, Butcher P D, Stoker N G, Bioinformatics 2003, 19(1):53-61). Throughput of the reaction may be biased by secondary and tertiary structures of mRNA, affinities specific to the reverse transcriptase, inhibitors present in the sample, priming strategy, and variation in priming efficiency (Stahlberg A, Hakansson J, Xian X J, Semb H, Kubista M, Clinical Chemistry 2004, 50(3):509-515). The most common priming strategies utilize oligo-dT primers, random primers, or gene-specific primers. When oligo-dT primers are used for reverse transcription, RNA secondary structure and variation in poly(A) tail length may result in gene amplification 3′ bias. Random primers, often used in prokaryotic systems, fail to discriminate between mRNA and the preponderance of RNA in the form of ribosomal (rRNA) or transfer RNA (tRNA). Random hexamers, the most commonly employed, amplify only fraction of the transcriptome, comparing with random pentadecamers (Stangegaard M, Dufva I H, Dufva M, BioTechniques 2006, 40(5):649-657). However, random oligonucleotides of any size also prime abundant rRNAs and tRNAs that can lead to high background and misleading signal.
Ribosomal RNA (rRNA) sequences in many prokaryotes are GC rich relative to the genome at large and are highly conserved. These properties have been used to design non-random hexamers (HD/DHTTTT) to prime reverse transcription reactions (Gonzalez J M, Robb F T, J Microbiol Methods 2007, 71(3):288-291). The result was a counter-selective synthesis of cDNA corresponding to mRNA from prokaryotic total RNA extractions. In contrast, application of gene-specific primers on a genomic scale requires synthesis of multiple primers. An algorithm to predict the minimal number of non-degenerate genome-directed primers that specifically anneal to all genes in a given genome has been designed and successfully applied in bacteria (Talaat A M, Hunter P, Johnston S A, Nat Biotechnol 2000, 18(6):679-682).
Another lately developed method relies on a collection of short, computationally selected oligonucleotides (‘not-so-random’ (NSR) primers) to obtain full-length, strand-specific representation of nonribosomal RNA transcripts (Armour C D, Castle J C, Chen R, Babak T, Loerch P, Jackson S, Shah J K, Dey J, Rohl C A, Johnson J M, Nature Methods 2009, 6(9):647-649). Selective enrichment of non-rRNA targets was achieved by computationally subtracting rRNA priming sequences from a random hexamer library. The presence of rRNA and tRNA plagues most mRNA purification procedures due to their relative abundance, leading to non-specific interactions like rRNA adsorption to the oligo-dT matrix, or hybridization of rRNA and mRNA sequences (Matveeva O M, Shabalina S A, Nucl Acids Res 1993, 21:1007-1007).
What is needed in the art is a method of amplifying and quantifying selected mRNA sequences, without interference from rRNA or tRNA. The present invention is believed to be an answer to that need.